Project Summary/Abstract Global incidence of Type 2 Diabetes (T2D) has reached epidemic proportions, and increasing evidence indicates that dysfunctional adipose tissue is an important contributor to the pathogenesis of T2D. Expanding catabolic adipocyte phenotypes within adipose tissues through adrenergic activation improves energy balance and insulin sensitivity. In order to exploit this remodeling of adipose tissue for therapeutic benefit, we need to understand the mechanisms by which adrenergic signaling expand the population of catabolic adipocytes in vivo. Brown adipose tissue (BAT) is a thermogenic organ and experimental activation of BAT in rodents improves metabolic profiles in models of T2D. BAT is also present in adult humans, where cold-induced activity is correlated with metabolic health. However, the amount of human BAT is highly variable and has low thermogenic activity. Therefore, in order to expand the thermogenic activity of BAT in humans, it is necessary to understand the source of new brown adipocytes and how they are recruited and maintained in adult mammals. BAT neogenesis is the principal means by which cold exposure expands thermogenic capacity, and thus cold-induced neogenesis provides a physiologically relevant model of brown adipogenesis in vivo. Previous work from our lab has demonstrated that brown adipogenesis occurs within a spatially defined tissue zone and involves the proliferation of adipocytes progenitors, tissue monocytes/macrophages, capillary endothelial cells, as well as an uncharacterized population of stromal cells. The overall goal of this proposal is to deconstruct the neogenic zone in adult BAT, and to test specific mechanisms by which adrenergic signaling induces BAT neogenesis. We proposed to use single cell RNA-sequencing (scRNA-seq) to deconstruct the cell types present in the brown neogenic zone in a comprehensive and unbiased fashion. Single cell profiling over the course of cold-induced neogenesis will define the cell types involved, and identify the differentiation trajectories of activated brown adipocyte progenitors in vivo. In addition, we will address specific mechanisms by which neural activation triggers tissue expansion using novel genetic loss-of-function analyses. The project will not only significantly advanced our understanding of the composition of brown adipose tissue and how beta adrenergic receptor activation leads to neogenesis in vivo, but will introduce new and innovative tools to the field, such as smFISH and scRNA-seq. The team of mentors, having highly complementary expertise, will provide an integrative, multidisciplinary training experience.